Free-fatty acid release from adipose tissue is the sole mechanism for the mobilization of fat cell triglyceride. Defective FFA release in obese subjects will result in the maintenance of large fat stores and the perpetuation of obesity. Excessive FFA release on the other hand might lead to increased lipoprotein formation or a worsening of reduced glucose disposal. We have used FFA turnover and lipid oxidation rate, assessment of body composition to measure total fat mass, and in vitro measurements of lipolytic rates in isolated fat cells, in the same subjects to investigate possible mechanisms of regulation of in vivo FFA metabolism. The data indicate that fatty acid availability or use per kg of fat actually decreases with increasing obesity. This could be due to a contribution of nonadipose tissue triglyceride to the FFA turnover and lipid oxidation. Even more likely, however, is that plasma or local tissue factors in obese individuals prevent fatty acid from becoming available in spite of increased stores. This was also suggested by the findings in vitro. These data suggested that the fat cells were more than capable of releasing large amounts of FFA in obese subjects but apparently failed to do so in vivo. These results indicate that in obese subjects much of the increased fat store may not be accessible to the rest of the body. Furthermore, in vitro measurements of fat cell lipolysis cannot be used to directly predict in vivo fatty acid metabolism. Finally, these data indicate that there is a large non-oxidative fatty acid disposal that may be important in the regulation of the plasma FFA concentration.